Research Project Updates

Nocturnin, a peripheral clock gene, regulates bone mass, marrow adiposity, and IGF-I in mice, Masanobu Kawai, MD, PhD.Rosiglitazone (ROZ) treatment of mesenchymal cells (MSCs) suppresses osteoblastogenic genes and increases adipogenic genes. Through microarray analysis we have identified nocturnin (NOC) as a gene that is altered in response to ROZ in MSCs. Noc is a peripheral clock gene encoding a deadenylase expressed in bone, fat, liver and stem cells. Hepatic Noc mRNA exhibits a circadian rhythm, being highest early at night. RT-PCR from MSCs showed increased Noc expression during adipogenesis but reduced expression during osteoblastogenesis. Thus, we hypothesized that Noc was involved in cell specification and was an important determinant of bone mass. Our experiments to date have demonstrated Noc has an important role in cell specification of MSCs, and may be regulating bone mass by modulating IGF-1 expression within the skeletal environment. Most recently we have shown that Noc acts as a chaperone for PParγ translocation from the cell membrane to the nucleus. This has implications for the circadian regulation of fat metabolism and potentially makes Noc an important therapeutic target for obesity.

IGFBP-2 regulates bone turnover likely through a mechanism which is independent of IGF-1 binding and involves activation of Wnt/β-catenin signaling, Masanobu Kawai, MD, PhD.We previously reported that Igfbp2 null mice (BP2-/- mice) have a skeletal phenotype that is characterized by reduced bone turnover and a low bone volume fraction. In addition, we noted that PTEN was increased in BP2-/- mice. In this study we showed that IGFBP2regulated bone remodeling independent of IGF-1 binding through modulation of the Wnt/β-catenin signaling pathway. Our experiments have shown the IGF-1 independent actions of IGFBP2 are mediated through the heparin binding domain. Activation of the heparin binding domain results in increased bone mass and therefore IGFBP2 may be a potential therapeutic target for the treatment of osteoporosis.

Fat specific PPAR deletion mice exhibit high bone mass phenotype before developing lipodystrophy, Masanobu Kawai, MD, PhD.Several lines of evidence have shown the negative relationship between bone and fat. For example, age-related bone loss has been shown to be associated with increased marrow adiposity. One underlying mechanism possibly resides in the activation of the PPAR2 gene in bone marrow since PPAR2 is increased in aging bone marrow and PPAR haploinsufficiency leads to increased bone mass in mice. To fully understand the role of PPAR in bone-fat interaction, we analyzed skeletal phenotypes using fat specific deletion of PPAR with an aP2-Cre lox P system (FKO�� mice). These mice have been reported to be insulin resistant due to lipodystrophy by 6 months of age. Our investigations have shown the FKO mice exhibit gender and compartment-specific skeletal and fat phenotypes; female FKO mice display high trabecular bone mass before developing lipodystrophy. Our data raise important questions about the bone-fat interaction and suggest there may be potential therapeutic targets for bone in fat tissue.

Bone Marrow Transplantation (BMT) Differentially Rescues Cortical but not Trabecular Bone in an Accelerated Aging Mouse Model Anne Breggia, PhD., IGFBP-2, a transport binding protein for IGF-1, is an essential factor for in vitro propagation of hematopoietic stem cells (HSCs). Global deletion of IGFBP-2 (BP2) results in an accelerated aging phenotype with very low bone turnover, decreased trabecular and cortical bone mass and obesity in male but not female mice. We hypothesized the decreased bone turnover present in male BP2-/- mice would be rescued by bone marrow transplantation (BMT) with BP2+/+ bone marrow donor cells. Our experiments have shownthat the cortical but not the trabecularskeleton of IGFBP-2 null mice is rescued by BMT. These data support the tenet that stromal cell production of IGFBP2 may be essential for skeletal development and maintenance.

Influence of Risperidone in Bone and Adipose Tissue Metabolism Ingrid DePaula, MD, PhD, Risperidone is an antipsychotic drug used widely in children and adults which causes adverse effects including hyperprolactinemia, bone loss, significant bodyweight gain and other metabolic disorders. Understanding mechanisms of action of Risperidoine is important to prevent unwanted side effects and to understand the relationship of hyperprolactemia to obesity and bone loss. In this project we treat mice with Risperidone by adding it to their diet. In our experiments to date, analysis of fat and bone metabolism in our experimental and control mice have shown that even though there was not increased total bodyweight gain, fat accumulated in the liver (fatty liver disease) and there was loss of cortical bone in the femur. These preliminary results reveal new insights into the relationship of bone and fat metabolism. In addition, our results also show new adverse effects which may need to be addressed when this drug is administered in the patient population, especially in children and adolescents. Further investigation of the metabolic effects of this drug will be important in uncovering other potential side effects and to institute preventive measures prior to Risperadone therapy.

Mechanisms of Parathyroid Hormone Action in Bone Francisco DePaula, MD, Parathyroid hormone (PTH) is the main hormone involved in the control of calcium metabolism and strongly influences the development and maintenance of bone mass. Its action on bone tissue is complex, being of a predominantly anabolic or catabolic nature. The factors that determine its action are not well understood, possibly involving changes in diurnal secretion such as basal secretion, number of pulses, pulse amplitude, and peptide fraction secreted. In addition, the pattern of response to PTH may depend on its interaction with 1,25(OH)2 D. Great advances have been made recently in the recognition of some factors that modulate the response of PTH to the bone microenvironment. However, we still do not know how excesses and deficiencies of 1,25(OH)2 D can interfere with this process. In the present proposal we intend to assess the influence of PTH treatment in VDR+/- mice to ascertain the evolution of bone mass under these circumstances. Our experiments to date have demonstrated that this animal model mimics a condition of modest vitamin D deficiency, most likely reflecting a frequent situation observed in aging and people living in higher latitude. This is clinically important as PTH is used in the treatment of osteoporosis. The study will also address the effects of vitamin D deficiency and PTH treatment on cardiac function.

Significance of the Absence of Brown Fat in the MISTY Mouse Strain and its Relationship to Bone Mass Masanobu Kawai, MD, PhD, Sheila Bornstein, MS., Our laboratory is interested in understanding the relationship of fat tissue to bone mass. In particular, we have been studying the role of brown fat, the type of adipose tissue that consumes oxygen and produces heat, in the acquisition of bone mass. The MISTY mice were initially discovered and used as a control strain for the obese, ob/ob mouse that has a deficiency of leptin. However, recent phenotyping of MISTY revealed the absence of brown fat even at an early age. Our interest in MISTY relates to the expression profile of bone and adipose tissue compared to control mice. More importantly, we have identified a gene, nocturnin, which is expressed early in fat differentiation, and may be essential for brown fat generation. Hence, we will be doing expression profiling in bone, fat and liver for brown fat genes and nocturnin and measuring bone mass at 4, 8, and 16 weeks of age. Our preliminary findings demonstrate a low bone mass and increased adipose tissue in the MISTY strain.

The Effect of Lipoxygenase 5 Deletion on Bone and Fat Metabolism Interactions Masanobu Kawai, MD, PhD, Phuong Le, MS, Lipoxygenase 5 null mice (Alox 5-/-) exhibit suppression of bone resorption. Our global hypothesis is that the relationship between marrow fat and bone formation is dependent on inter cellular signaling; hence, the absence of Alox 5- with reduced suppression of bone turnover, will have less marrow fat. Marrow fat is analyzed by two methods: MRI in vivo 2 weeks prior to sacrifice and the day of sacrifice, and histomorphometry of the distal femur with toludine blue staining for marrow adipocytes. Our preliminary findings with PIXIMUS demonstrate low bone mass for ALOX5-/- and an increase in percent fat in ALOX5 -/- on high fat diet.